Two forms of cytochrome P450 have been purified from bovine adrenal mitochondria to homogeneity and high specific activity: cytochromes P450scc (cholesterol side chain cleavage, CSCC) and P45011 Beta (steroid 11 Beta and 18-hydroxylation). Both cytochromes released core peptides containing heme when treated with BrCN. The purified peptides contained 41-45 amino acids. CSCC by P450scc was maximally active when cholesterol was added in 0.3% Tween 20. Tween both facilitated the availability of cholesterol to P450scc and stabilized the cytochrome. Complex formation between adrenodoxin and P450scc was studied by means of the spectral change upon complex formation. Complex formation was enhanced by cholesterol but-was not affected by Tween. Conversely, adrenodoxin enhanced cholesterol binding. The reaction product, pregnenolone, was competitively inhibited by adrenodoxin and cholesterol binding. The mechanism of electron transfer during CSCC was shown to involve transfer of reduced adrenodoxin from flavoprotein reductase to P450scc and to be critically determined by the proportion of reduced adrenodoxin as well as the total concentration. Cholesterol transport to P450scc in adrenal cells was shown to invlove two key steps: lipid droplet to mitochondria (sensitive to anti-microtubule and anti-microfilament agents) and intra-mitochondrial movement (sensitive to protein synthesis inhibitor). The kinetics of cholesterol transference to the mitochondria were measured in vivo and in isolated adrenal cells and were consistent with intra-mitochondrial transport being the slowest step. Examination of cholesterol distribution within the mitochondria indicated that protein synthesis inhibition prevents transfer of cholesterol from the outer membrane to the inner membrane where P450scc is located.